Acyl coenzyme A: cholesterol acyltransferase (ACAT) catalyzes the formation of intracellular cholesterol esters. It is present in a variety of tissues, and is believed to play significant roles in lipoprotein assembly and secretion, in steroid hormone production, and in dietary cholesterol absorption. Under pathological conditions, accumulation of ACAT reaction product as cytoplasmic cholesterol ester lipid droplets within macrophages and smooth muscle cells is a characteristic feature of early lesions of human atherosclerotic plaque. ACAT is a membrane protein located in the ER. The ACAT protein only exists in minute quantities. Its activity is susceptible to inactivation by detergents, and has never been purified to homogeneity. Through somatic cell and molecular genetic approach, we have recently succeeded in molecular cloning and functional expression of human macrophage ACAT cDNA K1. During the forthcoming period, our research will focus on pursuing five specific aims: A. To biochemically characterize human ACAT protein K1 which has been expressed in baculovirus infected insect cells. B. To define the mode(s) of regulation of human ACAT by cholesterol or 25- hydroxycholesterol in simple cell culture systems, including CHO cells expressing human ACAT activity, human fibroblasts, human liver tumor HepG2 cells, and phorbol ester activated human macrophage like THP-1 cells. C. To immunolocalize human ACAT protein in intact cells. D. To probe the molecular environment of ACAT protein K1 by chemical cross-linking, with the goal of isolating and identifying protein(s) tightly associated with ACAT protein K1 in the ER membrane. E. To characterize partial cDNA clone L1 as a candidate gene encoding a liver-specific human ACAT protein component. The outcome of this research will begin to fill a void in our understanding of the structure, function, and regulation of an enzyme that plays an important role in cholesterol metabolism, lipoprotein assembly, steroid hormone production, and atherogenesis.